KR102492243B1 - Homogeneous immunoassay with compensation for background signal - Google Patents

Abstract

The present invention relates to homogeneous immunoassays that allow for compensation of background signals inherent in samples and reagents. The present invention relates to the use of homologous immunoassays for the detection of the presence or amount of symmetrical dimethyl arginine (SDMA) in a biological sample. Reagents and kits for performing the assay.

Description

Homogeneous immunoassay with compensation for background signal

background

related application

This application claims the benefit of US Provisional Patent Application Serial No. 62/118,832, filed February 20, 2015, which is hereby incorporated by reference in its entirety.

Field

The present disclosure generally relates to homogeneous immunoassays that allow for compensation of background signals inherent in samples and reagents. In certain embodiments, the present disclosure relates to the use of homologous immunoassays for the detection of the presence or amount of symmetrical dimethyl arginine (SDMA) in a biological sample.

background

Homogeneous immunoassays have been implemented for the determination of various analytes, most notably, analytes for drugs of abuse. SDMA has been identified in biological samples as a marker for assessment of, for example, renal function, cardiovascular function, and SLE. SDMA is typically present in biological samples in relatively low concentrations compared to analytes for drugs of abuse.

Thus, the present inventors have identified a need in the art for a more accurate and sensitive allogeneic immunoassay, especially for analytes such as SDMA that have low concentrations in biological samples.

outline

In one aspect, the present disclosure relates to symmetrical conjugates of dimethyl arginine (SDMA), for example, conjugates of SDMA and an enzyme. In one example of the present disclosure, the conjugate is SDMA conjugated to glucose-6-phosphate dehydrogenase (G6PDH). In various embodiments, SDMA and enzyme are conjugated via a 5-15 member linker. Similarly, SDMA can be conjugated to the enzyme via a linker with a length of about 5-15 angstroms. Exemplary conjugates of the present disclosure include:

In another aspect, the disclosure relates to a composition comprising a conjugate of the disclosure and an antibody specific for free SDMA. In various embodiments, an antibody specific for free SDMA may have a reactivity to asymmetric dimethylarginine (ADMA) that is less than 25% of its reactivity to free SDMA. Similarly, the antibody may have no or substantially no cross-reactivity with one or more compounds selected from the group consisting of asymmetric dimethylarginine (ADMA), L-arginine, and N-methylarginine.

In a further aspect, the disclosure relates to a kit comprising a conjugate of the disclosure and an antibody specific for SDMA.

In various aspects, kits of the present disclosure include reagents for performing an assay on a sample containing an analyte. The kit may include the following components:

(a) i. A first reagent comprising an anti-analyte antibody and a signal generating substrate for the enzyme, and

ii. A first set of reagents for performing a first assay comprising a second reagent comprising a conjugate of an analyte and an enzyme, and

(b) i. A second set of reagents for performing a second assay comprising a third reagent comprising a substrate.

In the kit, the second set of reagents may further include a fourth reagent comprising at least one of a diluent and a buffer. The fourth reagent may also further include a conjugate or enzyme. In addition, at least one of the kit's reagents includes an inhibitor for an enzyme other than the enzyme of the conjugate. In an embodiment, the concentration of the conjugate of the first reagent is about 5 to about 150 times higher than the concentration of the conjugate of the fourth reagent. The kit may also include standards comprising a known amount of analyte diluted in a sample solution from which the analyte has been removed. For example, the analyte-depleted sample solution may be a depleted serum, a depleted plasma, or a pretreated sample. The first reagent and/or the second reagent may further include an electron mediator and a dye, the third reagent and/or the fourth reagent may further include a mediator and a dye, and the mediator receives electrons from the substrate. It transfers it to the dye.

Even further, the present disclosure relates to a reaction mixture comprising the components of the kit of the present disclosure and a sample suspected of containing SDMA.

In yet another embodiment, the present disclosure relates to a method for determining the presence or amount of free SDMA in a sample. The method comprises contacting a sample with an anti-SDMA antibody specific for free SDMA, the conjugate of claim 1, and a substrate comprising nicotinamide adenine dinucleotide (NAD), measuring the conversion of NAD to NADH, and measuring the conversion of NAD to NADH. and determining the presence or amount of SDMA in the sample based on the transition to

In various embodiments of the methods of the present disclosure, measuring conversion of NAD to NADH may include measuring conversion of NAD to NADH. For example, determining the presence or amount of SDMA in a sample based on NAD to NADH conversion may include comparing NAD to NADH conversion to a standard curve, or measuring NAD to NADH conversion may include NAD to NADH conversion. It may include measuring the amount converted to NADH in Determining the presence or amount of SDMA in a sample based on NAD to NADH conversion may also include comparing the amount of NAD to NADH conversion to a standard curve.

In another aspect of the method of the present disclosure, the method comprises:

(a) i. forming a sample first reaction mixture by contacting the sample with an anti-analyte antibody, a conjugate comprising an analyte and an enzyme, and a substrate that generates a signal upon contact with the enzyme;

ii. performing a sample first reaction sequence comprising measuring a signal from the sample first reaction mixture;

(b) i. forming a sample second reaction mixture by contacting the sample with a substrate;

ii. performing a sample second reaction sequence comprising measuring a signal from the sample second reaction mixture;

(c) subtracting the signal quantity from step (b) from the signal quantity from step (a) to give a net signal;

(d) using the net signal to determine the amount of analyte in the sample.

Methods of the present disclosure may further include the following steps:

(a) i. calibrators by individually contacting each calibrator of the set of calibrators containing a known amount of analyte with an anti-analyte antibody, a conjugate comprising the analyte and enzyme, and a substrate that generates a signal upon contact with the enzyme 1 to form a reaction mixture;

ii. performing a calibrator first reaction sequence comprising measuring a signal from each calibrator first reaction mixture;

(b) i. forming a calibrator second reaction mixture by contacting each calibrator of the set of calibrators with a substrate;

ii. performing a calibrator second reaction sequence comprising measuring a signal from the calibrator second reaction mixture;

(c) subtracting the signal quantity from step (a) from the signal quantity from step (b) to give a net signal for each calibrator;

(d) generating a standard curve using the net signals from at least two of the calibrators;

(e) determining the amount of analyte in the sample by comparing the net signal from the sample to a standard curve.

In another embodiment of the method of the present disclosure, the method may include the following steps:

(a) i. forming a sample first reaction mixture by contacting the sample with an anti-analyte antibody, a conjugate comprising an analyte and an enzyme, and a substrate that generates a signal upon contact with the enzyme;

ii. measuring a signal from the sample first reaction mixture;

iii. performing a sample first reaction sequence comprising normalizing reaction rates for the sample first reaction mixture to account for background associated with the sample first reaction mixture;

(b) i. forming a sample second reaction mixture by contacting the sample with a substrate;

ii. measuring a signal from the sample second reaction mixture;

iii. performing a sample second reaction sequence comprising normalizing a reaction rate from the sample second reaction mixture to account for a background associated with the sample second reaction mixture;

(c) subtracting the normalized rate of step (b)(iii) from the normalized rate of step (a)(iii) to provide a final reaction rate for the sample containing the analyte;

(d) determining the amount of analyte in the sample using the final reaction rate.

Methods of the present disclosure may also include the following steps:

(a) i. calibrators by individually contacting each calibrator of the set of calibrators containing a known amount of analyte with an anti-analyte antibody, a conjugate comprising the analyte and enzyme, and a substrate that generates a signal upon contact with the enzyme 1 to form a reaction mixture;

ii. measuring a signal from each calibrator first reaction mixture;

iii. performing a calibrator first reaction sequence comprising normalizing a reaction rate from each calibrator first reaction mixture to account for a background associated with each calibrator first reaction mixture;

(b) i. forming a calibrator second reaction mixture by contacting each calibrator of the set of calibrators with a substrate;

ii. measuring a signal from the calibrator second reaction mixture;

iii. performing a calibrator second reaction sequence comprising normalizing a reaction rate for each calibrator in the calibrator second reaction mixture to account for a background associated with the calibrator second reaction mixture;

(c) subtracting the normalized rate of step (b)(iii) from the normalized rate of step (a)(iii) to provide a final reaction rate for each calibrator;

(d) generating a normalized standard curve based on the final reaction rate for each calibrator;

(e) determining the amount of analyte in the sample by comparing the normalized reaction rate for the sample to a normalized standard curve.

In various aspects of the methods of the present disclosure, the sample and calibrator second reaction mixture may include any one or more of a diluent, a buffer, and an anti-analyte antibody. Further, consideration of the background associated with the sample first reaction mixture and/or calibrator first reaction mixture may include subtracting the background from each of a plurality of signal measurements associated with the determination of the reaction rate for each sample and/or calibrator first reaction mixture. can include Similarly, consideration of background associated with a sample and/or calibrator second reaction mixture may include subtracting background from each of a plurality of signal measurements associated with the determination of the reaction rate for the sample second reaction mixture and/or each calibrator second reaction mixture. include that

In the methods of the present disclosure, the sample may be a biological sample such as serum, plasma, urine or cerebrospinal fluid.

The method of claim 56 , wherein the calibrator comprises an analyte diluted in plasma or serum, including, for example, serum or plasma removed. In another embodiment, the calibrator may be a pretreated sample.

In a further aspect of the methods of the present disclosure, the sample and/or calibrator second reaction mixture may further comprise a conjugate. For example, the conjugate in the sample or calibrator first reaction mixture may be present in about 5 to about 150 times greater concentration of the conjugate in the sample or calibrator second reaction mixture.

Still further, in other aspects of the methods of the present disclosure, any reaction mixture may include an inhibitor for an enzyme other than the enzyme of the conjugate. In addition, any reaction mixture may further include an electron mediator and a dye, wherein the mediator accepts electrons from the substrate and transfers them to the dye.

In yet another aspect, the present disclosure relates to a method for determining chronic kidney disease in an animal. The method includes determining the presence or amount of SDMA in a biological sample from an animal according to the methods of the present disclosure, and determining chronic kidney disease in the animal based on the presence or amount of SDMA in the sample.

1 depicts the assay results of the present disclosure on human serum samples from a normal population and patients suffering from chronic kidney disease (CKD).
Figure 2 shows a schematic diagram of the procedure for conjugating SDMA to G6PDH using SIA to activate G6PDH.
Figure 3 shows a schematic diagram of the procedure for conjugating SDMA to G6PDH using SBAP to activate G6PDH.
Figure 4 shows a schematic diagram of the procedure for conjugating SDMA to G6PDH using SMCC to activate G6PDH.
Figure 5 is a schematic diagram of the mediator-dye reaction mechanism in which the mediator passes electrons to the dye to reduce the dye and shift the absorbance of the dye.
6 shows a calibration curve for SDMA spiked into human serum and analyzed according to the methods of the present disclosure.
7 shows calibration curves for SDMA spiked into human serum and analyzed according to the fixed calculation method and the rate calculation method of the present disclosure.
8 shows the results of an assay for a known concentration of SDMA performed according to the present disclosure without subtracting the background signal.
9, 10 and 11 show SDMA assay results according to the present disclosure using canine (FIG. 9), feline (FIG. 10), and equine (FIG. 11) ablated serum calibrators.
12 shows SDMA assay results according to the present disclosure using a buffer-based calibrator.

Described

Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise.

SDMA is symmetrical dimethylarginine. The structure of SDMA is as follows:

.

.

While one or more amino acid residues of SDMA may be present in a polypeptide, "free SDMA" refers to SDMA that is not part of a polypeptide chain, including salts of SDMA.

As used herein, the term "analog" generally refers to a modified form of an analyte that can compete with the analyte for a receptor, the modification being a means of linking the analyte to another moiety, such as a label or solid support. provides An analyte analog may bind an antibody in a manner similar to an analyte.

As used herein, the term “antibody” refers to a glycoprotein that is produced by B lymphoid cells, generally in response to exposure to an antigen, and that specifically binds to that antigen. The term "antibody" is used in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit the desired biological activity. antibodies), and antibody fragments.

As used herein, "anti-SDMA antibody," "anti-SDMA antibody portion," or "anti-SDMA antibody fragment" and/or "anti-SDMA antibody variant" and the like refer to, but are not limited to, the complementarity of one heavy or light chain constant region. Any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule, such as a critical region (CDR), framework region, or any portion thereof.

As used herein, the term “antibody fragment” refers to a portion of a full length antibody, usually an antigen binding or variable domain thereof. Specifically, for example, antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies from antibody fragments.

As used herein, the term "antigen" generally refers to a substance capable of reacting with an antibody specific for an antigen under appropriate conditions.

As used herein, the term "analyte" generally refers to a substance, or set of substances, in a sample that is to be detected and/or measured.

As used herein, the term "biological sample" refers to a tissue or fluid sample, generally from a human or animal, and includes, but is not limited to, whole blood, plasma, serum, spinal fluid, such as cerebrospinal fluid; samples of lymph fluid, peritoneal fluid (ascites), external sections of skin, respiratory, intestinal and genitourinary tract, tears, saliva, urine, blood cells, tumors, organs, tissues, and in vitro cell culture components.

As used herein, "cross-reactivity" generally refers to the ability of an individual antigen-binding site of an antibody to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. Generally, cross-reactivity occurs because (i) the cross-reacting antigen shares a common epitope with the immunizing antigen or (ii) it has an epitope structurally similar to that on the immunizing antigen (multispecificity).

As used herein, the term "label" refers to a detectable compound or composition that can be conjugated directly or indirectly (e.g., through covalent or non-covalent means, alone or encapsulated) to an analyte analog, such as an SDMA analog. For example, an enzymatic label can catalyze chemical alteration of a detectable substrate compound or composition. Enzymes utilized in the present disclosure include, but are not limited to, alkaline phosphatase (AP); glucose-6-phosphate dehydrogenase ("G6PDH"); beta galactosidase (B GAL); and horseradish peroxidase (HRP), malate dehydrogenase (MDH). Any description of an enzyme herein such as "G6PDH" or "glucose-6-phosphate dehydrogenase" also includes variants, isoforms and mutants of the enzyme, for example described in US 6,455,288, which is incorporated herein by reference in its entirety. G6PDH with amino acid substitutions. Utilization of the label can be detected by means such as direct visualization or detection of electromagnetic radiation, and optionally produces a measurable signal.

As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibody comprising populations are identical. Monoclonal antibodies are highly specific and directed against a single antigenic site. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" merely indicates the character of the antibody and is not to be construed as requiring production of the antibody by any particular method. Specifically, for example, monoclonal antibodies can be made by hybridoma methods, can be made by recombinant DNA methods, or can be isolated from phage antibody libraries using known techniques.

As used herein, the term “polypeptide” refers to a molecule having a sequence of amino acids linked generally by peptide bonds. The term includes proteins, fusion proteins, oligopeptides, cyclic peptides, and polypeptide derivatives. Although antibodies and antibody derivatives are discussed in separate sections above, antibodies and antibody derivatives are treated as a subclass of polypeptides and polypeptide derivatives for the purposes of this invention.

"Receptor" means any compound or composition capable of recognizing a specific spatial and polar organization of a molecule, such as an epitope or determinant site. Exemplary receptors include antibodies, Fab fragments, and the like.

"Binding specificity" or "specific binding" refers to a second molecule of a first molecule, e.g., an analyte such as SDMA, and a polyclonal or monoclonal antibody, or antibody fragment specific for an analyte (e.g., Fv , single-chain Fv, Fab', or F(ab) ₂ fragments). For example, as used herein, "specificity" generally refers to the ability of an individual antibody binding site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in analyte-antibody reactions. Antibodies are capable of discriminating differences in (i) primary structure of an analyte, (ii) isomeric forms of an analyte, and (iii) secondary and tertiary structures of an analyte, where applicable. Antibody-analyte reactions exhibiting high specificity exhibit low cross-reactivity.

"Substantial binding" or "substantially binds" refers to the degree of specific binding or recognition between molecules of an assay mixture under specific assay conditions. In its broadest aspect, substantial binding relates to the difference between a first molecule's inability to bind to or recognize a second molecule and a first molecule's ability to bind to or recognize a third molecule. and whereby the difference is sufficient to allow a meaningful assay to be performed that distinguishes specific binding under a specific set of assay conditions, including the relative concentrations of the molecules and the time and temperature of incubation. In another embodiment, one molecule is unable to substantially bind to or recognize another molecule in a cross-reactive sense, wherein the first molecule exhibits less than 25% of the reactivity to a third molecule under a particular set of assay conditions, e.g. eg less than 20%, less than 15%, less than 10%, less than 5% or less than 1% reactivity towards the second molecule. Specific binding can be tested using a number of well-known methods such as immunohistochemical assays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or Western blot assays.

As used herein, the term "salt" refers to a salt formed between an acid and a basic functional group of a compound. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid sheet Lates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, genticinates, fumarates, gluconates, glucaronates, saccharates, formates, benzos ates, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) contains salt. The term "salt" also refers to a salt formed between a compound having an acidic functional group, such as a carboxylic acid functional group, and an inorganic or organic base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines) such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amine such as N,N,-dimethyl-N-(2-hydroxyethyl)amine , or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

Turning now to the present disclosure, in general, the present disclosure directs the immunodetection of an analyte in a sample in a manner that accounts for the background signal associated with the sample and reagent components other than the analyte. For example, components of a sample other than an analyte may react with each other or with components of a reagent for an assay. Such reactions may interfere with the accuracy of the detection method. Biological samples are notorious for containing many components that can create background noise in assays.

Samples can be analyzed using a modified assay based on the Enzyme Multiplied Immunoassay Technique (EMIT®) homologous immunoassay system. In the traditional EMIT® assay, a sample containing an analyte is contacted with an anti-analyte antibody, a conjugate of the analyte and enzyme, and a substrate that produces a signal upon contact with the enzyme. Binding of the antibody to the conjugate inhibits or reduces enzymatic activity. When an analyte is present in the sample, the sample analyte competes with the conjugated analyte for binding to the antibody, which results in the production of more signal from the enzyme/substrate. In the absence of an analyte, more binding may occur between the antibody and the conjugate, limiting or preventing signal generation. Thus, more signal is produced when more analyte is present. Kinetic assays can use the rate of signal generation as an indicator of the presence or amount of an analyte in a sample.

In one aspect of the present disclosure, the traditional EMIT® assay format is performed with a first sequence of reactions for both the analyte and the calibrator. For convenience, this first reaction sequence may be identified herein as the "Color Assay". Then, in a modified assay of the present disclosure, a second and separate assay is performed with a second sequence of reactions for the sample and calibrator. For convenience, the second sequence of reactions may be identified herein as a “Blank Assay”. Reagents in blank assays do not contain anti-analyte antibodies and typically do not contain conjugates. Thus, the blank assay provides a sample-dependent background signal.

In various aspects, the present disclosure relates to the use of signals in two exemplary methods to determine the concentration of an analyte. For convenience, the exemplary methods are referred to herein as the "Rate" method and the "Fixed" method. In both methods, the difference between the signal or amount of signals in the color and blank assays is used. In one embodiment, the signal is measured as absorbance at a wavelength specific to the enzyme/substrate system, as is well known in the art. For example, for a G6PDH/NAD enzyme/substrate system, measurement of the absorbance at 340 nm will provide a value for the relative amount of NAD to NADH conversion in the presence of G6PDH. This value can be used to give either a net signal (for stationary methods) or a rate of reaction (for kinetic methods) reflecting the conversion of the substrate by the enzyme. Alternatively, enzymatic substrate reactions can be employed with electron carriers that mediate electron transfer between the substrate and various electron acceptors (e.g., dyes) that will allow measurement of reaction rates at wavelengths different from those of the substrate. .

In the fixed method, the net signal is computed. In one embodiment, the net signal is based on the difference in signal (eg, absorbance) between the color and blank assays at a given endpoint, which may or may not be reaction completion by depletion of all substrates. this is all The amount of difference in signal measured at the endpoints of the blank and color assays can be used to provide the net signal (eg, net signal = [amount of color signal] - [amount of blank signal]). Alternatively, the difference in the amount of signal between a given starting point (T1) (which may be at the completion of the combination of all reagents or some other predetermined time thereafter) and a given end point (T2) is the color and blank assay can be determined for both. The difference in the amount of signal from these decisions can then be used to determine the net signal (e.g., net signal = ([T2 color] - [T1 color]) - ([T2 blank] - [T1 blank)) . The net signal can be compared to a calibration curve to determine the amount of analyte in the sample.

When a net single is calculated based on a single measurement from each color and blank assay, measurements can be made after sufficient time has passed for reagents to react. For example, a single measurement in each assay can be made about 30 seconds to about 10 minutes after combining all reagents. More specifically, a single measurement is 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570 and 600 after a combination of all reagents. This can be done in one second. When two signals are measured in each blank and color assay, the first measurement (T1) is made approximately 15 seconds to 2 minutes after the start of the assay (combination of sample and all reagents). This time may be adjusted based on the concentration of the reagent and the expected concentration of the sample. In particular, T1 may be 15, 30, 45, 60, 75, 90, 105 or 120 seconds after combining all reagents. A second measurement (T2) may be made 15 seconds to several minutes after T1. For example, T2 may be, for example, 15 seconds, 18 seconds, 30 seconds, 45 seconds, or 1, 2, 3, 4, or 5 minutes after the first measurement T1.

In the rate method, the signal from the blank assay is subtracted from the signal from the color assay at defined intervals to give the reaction rate. The kinetics of the calibrator are used to provide a calibration curve, which can be used to determine the amount of analyte in the sample by comparing the kinetics curve of the assay for the sample with the calibration curve.

In a kinetic method, the rate of reaction can be determined by measuring the signal (eg, absorbance) at multiple time points during an enzyme mediated reaction. Determination of the time and interval of signal measurement is within the skill of the art given the reagent concentration and assay temperature. For example, the rate can be determined by measuring the absorbance starting about 2-10 minutes after the sample (or calibrator) and all reagents are combined at room temperature, taking measurements every 5-60 seconds for an additional 1-15 minutes. Reaction rate can be expressed as the change in absorbance over time. For example, absorbance can be measured starting about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes after combining the sample (or calibrator) and all reagents. Absorbance is typically measured for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes over 5, 10, 15, 20, 25, 30, 35, 40, Measured at intervals of 45, 50, 55 or 60 seconds. Each of these times may be extended or shortened depending on reaction conditions, analytes, and reagents.

In either fixed or rate method, the background from the sample first reaction mixture can be taken into account in the calculation of the net signal or reaction rate to provide a normalized signal. In one embodiment, the background absorbance is measured from a calibrator matrix used in place of the sample. The absorbance of the calibrator matrix is initially measured upon combination of all reagents and the matrix, or at a defined time thereafter. The calibrator matrix may be a calibrator or control mixture lacking any analyte. For example, a calibrator matrix lacking an analyte may be a sample from which the analyte has been removed by dialysis or by other pretreatment processes described further herein. In one embodiment, removed or endogenous species-specific or non-specific serum, plasma, or other biological fluid can be used as the calibrator matrix. In other embodiments described further herein, the calibrator matrix can be water or diluent containing proteins and/or other compositions (eg, BSA in PBS). A calibrator matrix is used in place of samples in both assays to provide a background signal for both the color assay and the blank assay.

Once the background has been determined, the background is then subtracted from the net signal (or measurements at T1 and T2) in the fixed method or from each measurement used to determine the sample kinetics in the rate method. For example, in the rate method, the absorbance of the calibrator matrix (with reagents) is subtracted from each absorbance measurement used to determine the rate of reaction for the sample (ie, the change in absorbance over time). In another aspect, the background rate is determined in a manner similar to the sample reaction rate. The background rate is then subtracted from the sample reaction rate to provide the normalized reaction rate for the first sample reaction mixture.

Accordingly, the present disclosure relates to methods of performing assays for analytes. The method performs a color assay on a sample by forming a sample first reaction mixture comprising the sample, an anti-analyte antibody, a conjugate comprising an analyte and an enzyme, and a substrate that generates a signal upon contact with the enzyme. (sample first reaction sequence). Typically, a sample first reaction mixture is formed by first contacting the sample with the antibody and substrate and then adding the conjugate in a second step. However, the sample may first be combined with the antibody and conjugate, and a substrate may be added in a second step. In one embodiment, the enzyme and conjugate are kept separate until the reaction sequence is ready to begin. As used herein, "contacting", in its broadest form, is used herein to refer to combining reagents in any order unless otherwise specified. Once a sample first reaction mixture has formed, the amount of signal can be measured from the mixture immediately or at one or more predetermined times thereafter for use in a fixed or rate method of the present disclosure.

In the modified fixed-and-rate method of the present disclosure, a separate assay sequence (sample second reaction sequence or "blank assay") is performed in a manner similar to the sample first reaction sequence. However, the reagents in the sample second reaction sequence do not contain anti-analyte antibodies. Typically, reagents in blank assays do not contain conjugates, but embodiments in which small amounts of conjugates or enzymes are added are described herein. In particular, a sample second reaction mixture is formed by contacting the sample with a conjugate that is free of a substrate and, in some cases, an anti-analyte antibody. A signal is measured from the sample second reaction mixture. The net signal or rate using the background from the calibrator matrix is determined in the same way as the sample first reaction sequence to provide a normalized net signal or normalized reaction rate for the sample second reaction sequence.

In the rate method, the normalized rate of the sample second reaction sequence is subtracted from the normalized reaction rate of the sample first reaction sequence to give the final reaction rate for the sample. The final reaction rate can be used to determine the presence or amount of an analyte in a sample. Typically, this is done by comparing the final reaction rate for a sample to a standard curve that can be prepared according to methods known in the art.

Color and blank assays for calibrators can be performed in a similar manner to color and blank assays for samples. Thus, a series of calibrator first reaction sequences (color assays) is performed by contacting each calibrator of a set of calibrators containing a known amount of analyte with an anti-analyte antibody, a conjugate comprising analyte and enzyme, and an enzyme. by forming a calibrator first reaction mixture by individually contacting it with a substrate that produces a signal. The signal is measured from each of the calibrator first reaction mixture and the normalized net signal (fixed method) or the normalized reaction rate for each of the calibrator first reaction mixtures (rate method) is determined from the calibrator first reaction mixture in the manner described above for the sample first reaction mixture. 1 is created taking into account the background associated with each of the reaction mixtures. One of the calibrators may be a matrix of calibrators (which does not contain an analyte) used to determine the background.

In a separate reaction series (blank assay), the calibrator second reaction sequence contacts each calibrator of the calibrator set with the anti-analyte antibody and, typically, the substrate in the absence of the conjugate, thereby forming a calibrator second reaction mixture. done by doing In some cases, the conjugate may also be present in minor amounts, generally as further described herein. A signal is measured for each of the calibrator second reaction mixtures, and the net signal or reaction rate for each mixture is normalized to account for the background associated with the calibrator second reaction mixture in the manner described above.

Thus, to account for the background signal in the fixed method, the normalized net signal from the blank assay is subtracted from the normalized net signal from the color assay. In the rate method, the final reaction rate for each calibrator is determined by subtracting the normalized rate from the calibrator in the calibrator second response sequence from the normalized rate for each calibrator in the calibrator first response sequence. Then, a normalized standard curve is created based on the final reaction rate for each calibrator, and the amount of the analyte in the sample can be determined by comparing the normalized reaction rate for the sample with the standardized standard curve.

In one embodiment, the reagents associated with the color assay and blank assay are shown in Table 1. Reagents are combined in appropriate diluents and/or buffers. As shown in Table 1, Blank Assay Reagent 2 (R2) does not contain conjugate, but it may be present as further described herein to ensure that the reaction has an appropriate volume of buffer. In some embodiments, blank assay R1 may have extra volume if blank assay R2 is not used. In one embodiment, the reagents for the color assay are the same as those for the blank assay except for the presence or absence of antibody and conjugate. When R2 does not contain any conjugates in blank assays, it still contains diluents, buffers and other components of R2 for color assays.

Table 1

In some embodiments, blank assay R1 may also contain an anti-analyte antibody.

Calibrators and test samples in color assays can generally be expected to result in an increase in absorbance due to the analyte-enzyme conjugate added to the reaction mixture. Many samples also give a positive reaction when tested in the blank assay, since the endogenous substrate or the endogenous enzyme reactive with the substrate of R1 will result in an increase in absorbance even in the absence of the conjugate. Often, however, samples and calibrators are diluted to such an extent that the background signal, usually small, is further attenuated so that the reaction mixture changes little to no absorbance when tested in a blank assay. Since the change in absorbance can fall below the sensitivity range of the detector, it can lead to calibration curve failure on the automated analyzer.

To avoid this problem, enzymes or analyte-enzyme conjugates can be added to the blank assay while ensuring that small positive changes in absorbance do not occur (regardless of calibrator or sample behavior). Accordingly, Table 2 presents the reagents present in this embodiment.

Table 2

The concentration of the enzyme or conjugate in R2 is typically kept low to avoid unwanted noise in the assay on the calibrator matrix. For example, when the enzyme is G6PDH, about 1-15 ng/mL of enzyme (conjugated or unconjugated) can be added to the blank assay. In particular, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng/ml may be added. The amount of conjugate in R2 is dependent on the size of the analyte, since a larger analyte will require a larger amount of conjugate to provide the same amount of enzyme. In one embodiment, the enzyme concentration (either alone or conjugated) in R2 for the color assay is about 10 to about 150 fold higher than the enzyme concentration in R2 for the blank assay. In various embodiments, the concentration of the conjugate in the color assay is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 times higher. When conjugated, the activity of the enzyme is typically lower than that of the unconjugated enzyme. For example, the activity of the conjugated enzyme is significantly different from that of the unconjugated enzyme, for example, the activity of the conjugated enzyme is 10%, 20%, 30%, 40%, 50% of the unconjugated enzyme. %, 60%, 70%, 80% or 90%. Thus, when an enzyme not conjugated to R2 for the blank assay is used, it can be used in a smaller amount than when it was conjugated and still provide the same activity as the conjugated enzyme.

Substrates, or when an enzyme has more than one substrate, may be present in excess of the reagent.

In some embodiments, an assay reagent may include a stabilizer of the assay reagent. For example, when the signal generating enzyme is glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate (G6P), or NAD, can be added to the conjugate-containing reagent to stabilize the enzyme-analyte conjugate. can

Serum contains several enzymes that may contribute to the background. Thus, addition of specific enzyme inhibitors to the reagent diluent can further improve assay accuracy. Suppressors help remove some of the interfering noise in color and blank assays. Thus, in various embodiments, a reaction component may include an inhibitor of an endogenous sample enzyme. For example, sodium oxamate is a known inhibitor of lactate dehydrogenase (LDH), an enzyme of NAD and/or NADP. Sodium oxamate prevents background signals associated with endogenous sample LDH by preventing LDH from turning over NAD in the sample or NAD, which may be part of the enzyme-substrate system associated with the assay (eg, G6PDH/NAD). More than 30 serum enzyme and enzyme inhibitor combinations are known and inhibitors are available for many of them.

In one embodiment, the calibrator contains a known amount of analyte (or none in the case of a calibrator matrix) in deionized or saline water to provide a calibration matrix that is used in place of the sample. In another embodiment, removed serum is used instead of water or saline. Dialyzed serum is, for example, native species-specific or non-specific serum or plasma from which an analyte has been removed in a dialysis process. A number of different sample preparation processes are known to remove or inactivate analytes. In each of these embodiments, the solution is spiked with a known amount of analyte to provide a series of calibrators over a range of expected analyte concentrations in the sample. In yet another embodiment, the calibration solution is a protein based solution such as 1-7% BSA in PBS. In this embodiment, it may be necessary to calibrate the BSA calibrator against the removed and spiked serum calibrator.

Typically, blank assays are intended to compensate for endogenous proteins, enzymes, or other large molecular components contributing to the background signal, although in many cases sufficient specificity can be achieved by the use of water or saline as a calibrator solution for biological samples. Although it can be obtained, water or saline is more suitable for non-biological samples. In addition, the use of water or saline buffers in the calibrator matrices of automated analyzers can tend to generate error messages for the calibrator matrices as the reaction rate associated with the use of buffers can be significantly increased compared to serum. In this situation, negative velocity could be generated by subtracting the absorbance values of the calibration matrix. These rates can be standardized for serum-based calibrator matrices, but automated assays will typically generate error messages in these situations.

According to one embodiment of the present disclosure, the analyte is SDMA and the enzyme-conjugate system is G6PDH/NAD. In this embodiment, an analog of SDMA is conjugated to G6PDH and used as a conjugate in a color and blank assay to determine the presence or amount of SDMA in serum or plasma samples from animals such as humans, cats and dogs. In one aspect of this embodiment, a calibrator is prepared by combining a known amount of SDMA with a calibrator matrix (depleted serum or plasma). Color and blank assays are performed as described above using the following reagents in exemplary amounts listed in Table 3:

Table 3

In particular, the anti-SDMA antibody may have a concentration in R1 of the color assay of from about 4 to about 10 μg/mL, eg about 4, 5, 6, 7, 8, 9 or 10 μg/mL. In R1 of the color assay or blank assay, G6P and NAD are about 8-75 mM, more particularly about 10-65 mM, or about 20-55 mM, and more particularly about 8, 10, 15, 20, 25, 30, It may have a concentration of 35, 40, 45, 50, 55, 60, 65, or 70 mM. In R2 of the color assay, the SDMA-G6PHD conjugate will have a concentration of about 0.25 to about 0.75 μg/mL, more particularly about 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.65, 0.70 or 0.75 μg/mL. can In R2 of the blank assay, the concentration of the conjugate may be about 5-30 ng/mL, more particularly about 5, 10, 15, 20, 25 or 30 ng/mL.

G6P is present in the R2 reagent to stabilize the enzyme. Alternatively, NAD could be used. Stabilizing the substrate is optional.

Reagents can be combined with the following volumes of samples and calibrators for color and blank assays, but volumes can be adjusted to accommodate different analytes and reaction conditions:

Sample: 5-20 μL

R1: 20-60 μl

R2: 20-150 µL

In one particular embodiment, the reaction mixture contains 10 μl of sample, 40 μl of R1 and 125 μl of R2.

In color and blank assays, these reagents can be used to create a calibration curve, which can be used to determine the presence or amount of SDMA in a serum or plasma sample. Figure 1 shows the results using the rate method for the determination of SDMA in normal and chronic kidney disease human serum samples by Beckman AU680® clinical chemistry analyzer. Results show accurate determination of SDMA concentration compared to gold-standard LC-MS values. Human serum (not stripped) was used as a calibrator matrix. Accordingly, the present disclosure relates to methods for determining chronic kidney disease in humans and animals including, for example, domestic, farm, and zoo animals. The method determines the concentration of SDMA in a biological sample from an animal subject according to the methods of the present disclosure, and the concentration is calculated using a standard curve or the amount of SDMA in a sample from the same species as the subject in healthy subjects and subjects suffering from chronic kidney disease. including comparison with other models with

In one embodiment, the disclosure relates to a kit containing reagents for determining the presence or amount of an analyte in a sample. For example, the kit may include a first set of reagents for performing a color assay, such as a first reagent comprising an anti-analyte antibody and a signal generating substrate for an enzyme, and a reagent comprising a conjugate of the analyte and enzyme. 2 may contain reagents. The kit may also include a second set of reagents including a third reagent containing a substrate. The second set of reagents may also include a fourth reagent containing a buffer/diluent, and optionally a conjugate. The purpose of the fourth reagent, containing only a buffer/diluent, is to ensure that the reaction involving the second set of reagents is carried out in the proper volume. When the second set of reagents does not include the fourth reagent, the volume of the third reagent should be adjusted accordingly. Additionally, the third or fourth reagent may contain an anti-analyte antibody.

The concentrations and amounts of the components in each reagent may be as described above for R1 and R2 in the color and blank assays, respectively. For example, the concentration of the conjugate in the first reagent is about 5 to about 150 times higher than the concentration of the conjugate in the fourth reagent. In one embodiment, at least one of the kit's reagents includes an inhibitor for an enzyme other than the enzyme of the conjugate. The kit may also include a standard or set of standards (calibrators) comprising a known amount of analyte diluted in an appropriate diluent such as water, saline, or removed or treated serum or plasma (eg, pretreated sample). there is.

When the analyte is SDMA, an exemplary kit includes an anti-SDMA antibody (polyclonal or monoclonal) and substrate(s) such as NAD and G6P in a first reagent. The second reagent includes a conjugate of G6PDH and an SDMA analogue as a stabilizer for the enzyme. The third reagent includes the SDMA-G6PDH conjugate and, optionally, the fourth reagent. The fourth reagent may contain only a diluent or buffer, or may contain a conjugate.

In addition to the kit, the present disclosure relates to a reaction mixture comprising reagents from the kit and a sample suspected of containing SDMA. For example, a reaction mixture can include a sample or calibrator, anti-SDMA antibody, SDMA-G6PDH conjugate, NAD, and G6P.

In each kit, method and reaction mixture of the present disclosure, a number of enzyme/substrate systems can be used in place of G6PDH/NAD. As another example, malate dehydrogenase is an enzyme that catalyzes the oxidation of malate to oxaloacetate using reduction of NAD+ to NADH similar to G6PDH. In addition, many enzyme mutants are known that enhance the signaling or stability of the enzyme. See, eg, US Pat. No. 6,455,288.

Analyte-enzyme conjugates can be prepared by a variety of known methods. The method and choice of reagents employed can provide linkers of varying lengths between the analyte and the enzyme. Linker length can affect the ability of the antibody to inhibit enzymatic activity and thus affect assay sensitivity. Typically, linkers of about 5-15 atoms, or 2-20 Angstroms, may be used. Conjugation of enzymes to analytes is within the skill of the art for many analytes that can be detected according to the present disclosure.

For example, FIG. 2 depicts conjugation of an SDMA analog of Formula 1 (below) to glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is activated with succinimidyl iodoacetate (SIA) prior to conjugation. Activation by SIA generates a five-atom linker between SDMA and the enzyme (-C-C-S-C-C(O)-), which does not contain unnatural nitrogen on SDMA resulting from replacing oxygen with nitrogen during derivatization of SDMA.

Figure 3 shows succinimidyl 3-(bromoacetamido)propionate (SBPA) resulting in a 9 member linker (not counting unnatural nitrogen) (-C-C-S-C-C(O)-C-N-C-C(O)-). Conjugation of the SDMA analog of Formula 1 to G6PDH using

Figure 4 shows conjugation of the SDMA analog of formula 1 to G6PDH using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) resulting in a 12 member linker. do.

The linker length can be adjusted by modifying G6PDH with other reagents or by using other SDMA analogs in the conjugation reaction, e.g., from about 5 to about 15 atoms, particularly about 5, 6, 7, 8, 9, 10 , linker lengths of 11, 12, 13, 14 or 15 atoms. Thus, the present disclosure is directed to methods 5-15 that include only atoms directly on the chain of the linker via the shortest path, excluding any atom on the side chain, substituent, or ring of the linker molecule that is not part of the shortest path between SDMA and the enzyme. It relates to a conjugate of SDMA and an enzyme through a linker of two atoms.

The linker length may range from about 2 Angstroms to about 20 Angstroms, particularly from about 5 to about 15 Angstroms, and more particularly from about 6-10 Angstroms.

In one embodiment, SDMA is conjugated to G6PDH in the presence of the G6PDH substrates glucose-6-phosphate (G6P) and/or nicotinamide adenine dinucleotide (NADH). Optimal conjugate-enzyme activity and inhibition of that activity by the antibody can be obtained by adjusting the ratios of enzyme, substrate and SDMA.

Several analogues of SDMA suitable for conjugation to G6PDH are described in US Pat. No. 8,481,690, incorporated herein by reference in its entirety. Depending on the desired length of the linker between SDMA and G6PDH, one of the following analogs may be used:

In the above formula, x and y are integers ranging from 1 to 5.

Formulas A, B and C provide available thiols that can react with conjugation targets that contain appropriate “thiol-reactive sites,” i.e., sites that will react with thiol groups. For example, maleimides, alkyl and aryl halides, and alpha-haloacyls are exemplary thiol-reactive moieties that can react with thiols to form thio-ethers. Similarly, pyridyl disulfides can react with thiols to form mixed disulfides. G6PDH activated with SIA, SBAP, or SMCC provides suitable thiol-reactive sites. When X=1, conjugation of Formula A with SIA-activated G6PDH provides a 5 atom linker between SDMA and G6PDH. Conjugation of Formula A (X=1) with SMCC results in a 12 member linker. Other linker lengths can be obtained by changing X.

In one particular embodiment where X=1, the SDMA analog has the formula (Formula 1):

Formula 1 can be prepared from SDMA (available from EMD Chemicals Inc. of Gibbstown, NJ) by the following exemplary synthetic scheme (1):

Schematic 1:

The primary and secondary amino groups of SDMA are protected by reacting SDMA with di-tert-butyldicarbonate (Boc ₂ O). The resulting tert-butoxycarbonyl (BOC) protected SDMA ((Boc ₃ )-SDMA, 1) is then bonded to the resin. For example, (Boc ₃ )-SDMA (1) is prepared by 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uranium hexane in dimethyl formamide (DMF). ₍ Boc ₃ )-SDMA (1) can be coupled to cysteamine-4-methoxy trityl resin (EMD Chemicals, Inc., Gibbstown, NJ). The BOC protecting group on the resin-bound (Boc ₃ )-SDMA cystamide (2) is removed and the resulting resin-bound SDMA cystamide is cleaved from the resin using, for example, trifluoroacetic acid in dichloromethane to form SDMA Cystamide (3) is provided, which is converted to the hydrochloride salt (4) by reaction with hydrochloric acid.

The traditional EMIT® assay measures the accumulation of NADH (or NADPH) by monitoring absorbance at 340 nm. In another embodiment of the present disclosure, adding a color changing dye and electron mediator to a reagent allows absorbance to be measured at absorbances other than 340 nm. In one specific example, the dye is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the vehicle is 1-methoxy phenazine methosulfate (PMS) . As depicted in Figure 5, PMS takes electrons from NADP and transfers them to MTT, which reduces MTT to give absorbance at about 650 nm.

Anti-SDMA antibodies can be polyclonal or monoclonal as described in US Pat. No. 8,481,690. Methods of producing polyclonal and monoclonal antibodies are within the skill of the art. In one embodiment, the antibody is a monoclonal antibody raised against the SDMA-KLH conjugate having the structure:

In various embodiments, the anti-SDMA antibody used in the modified assay of the present disclosure may have high specificity for SDMA and is one selected from the group consisting of asymmetric dimethylarginine (ADMA), L-arginine, and N-methylarginine. It may or may not have substantially no cross-reactivity with the above compounds. For example, an anti-SDMA antibody may have a reactivity of less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the reactivity exhibited for SDMA under a particular set of assay conditions, to ADMA, It is shown for L-arginine, and N-methylarginine.

All of the components described above can be used in a method for determining the presence or amount of free SDMA in a sample. For example, the method includes contacting the sample with an anti-SDMA antibody, a conjugate comprising SDMA and G6PDH, and a substrate(s) comprising NAD and G6P. NAD to NADH conversion is measured and compared to a standard curve to determine the presence or amount of SDMA in the sample as described herein. SDMA is conjugated to G6PDH by a 5-15 atom linker (2-10 angstroms) as described herein. Anti-SDMA antibodies can be monoclonal antibodies that have no or substantially no reactivity to ADMA, L-arginine, and N-methylarginine.

In yet another embodiment, the present disclosure is directed to a method for determining SDMA in a sample comprising derivatizing free SDMA and using an antibody directed against the derivative. For example, US Patent Publication 2004/0214252, incorporated herein by reference in its entirety, describes methods for modifying the guanidino nitrogen of SDMA and antibodies directed to modified SDMA. Thus, in one embodiment, the SDMA in the sample is modified prior to determination in a method according to the disclosure herein. In this embodiment, the anti-SDMA antibody must bind with sufficient affinity both the modified SDMA and the SDMA of the conjugate, which may also be modified, to provide a suitable assay.

Example

Example 1: Preparation of removed serum

Untreated commercial dog serum (500 mL) was loaded into bipedal SNAKESKIN™ dialysis tubes (3.5 K MWCO, 35 mm Dry I.D.) (Thermo Scientific) and incubated at least at 4°C against PBS buffer (20 L) with 20 g carbon powder. dialyzed for 6 hours. The process was repeated three times by exchanging the buffer and carbon.

SDMA concentrations in serum were measured by LC/MS before and after dialysis. In serum before dialysis, SDMA was 9.89 μg/dL. After dialysis, SDMA was 0.02 μg/dL.

Charred stripped dog serum was stored at -80°C for use.

Example 2: SDMA standard preparation:

SDMA.HCl (ChemBio) was dissolved in deionized water to a final concentration of 1000 μg/ml of SDMA. 200 μl of SDMA aqueous solution was added to 10 ml of stripped serum for a final stock solution with an SDMA concentration of 20 μg/ml. The solution was stored at -80 °C. A standard SDMA of 56 μg/dL was prepared by transferring 280 μl of SDMA stock solution to 10 ml of stripped serum. Other SDMA standards were prepared by serially diluting the stock solution in stripped serum to give solutions of 28.0, 14.0, 7.0, and 3.75 μl/dL. Standards can be verified by LC/MS.

Example 3: Preparation of Diluent

Diluent Formulations R1 and R2 contained the components identified in Table 4. Sodium oxamate is an optional component as otherwise described herein.

Table 4

Diluent Preparation Protocol

R1 Diluent (1 L scale). The following components were added to 500 ml of deionized water:

10 g BSA

50 ml 1M TRIS, pH8.0

0.372 g EDTA Sodium

4 ml of Brij-35 (30%)

4 ml of Proclin 150

0.96 g of PEG 6000

17.6 g of NaCl

10 ml mouse serum

1.665 g sodium oxamate (optional)

The solution was mixed well using a slow speed stir bar. When the powder was completely dissolved, the pH was adjusted to 7.0 with 10N NaOH or 3N HCl as needed. The solution was added to a graduated cylinder and made up to a final volume of 1 L with deionized water. After mixing well, the solution was gently mixed well, filtered through a 0.2 μm cellulose nitride filter unit and stored at 4° C. before use.

R2 Diluent (1 L scale): The following components were added to 500 ml of deionized water:

10 g BSA

100 ml 1M TRIS, pH8.0

2 ml 0.5M EDTA (pH8.0)

4 ml of Brij-35 (30%)

4 ml of Proclin 150

0.96 g of PEG 6000

17.6 g of NaCl

10 ml mouse serum

G6P 0.56g

1.665 g sodium oxamate (optional)

The solution was mixed well using a slow speed stir bar. When the powder was completely dissolved, the pH was adjusted to 8.0 with 10N NaOH or 3N HCl as needed. The solution was added to a graduated cylinder and made up to a final volume of 1 L with deionized water. After mixing well, the solution was gently mixed well, filtered through a 0.2 μm cellulose nitride filter unit and stored at 4° C. before use.

Example 4: Preparation of assay reagents

Preparation of R1 reagent and R1 blank

R1 reagent and R1 blank contained NAD (35 mM), G6P (56 mM) in R1 diluent. R1 reagent also included antibody (10.5 μg/ml). G6P is a stabilizer for the conjugate and is optional.

Diluents and other materials were brought to room temperature before preparing the reagents. A 2X substrate working solution (100 ml) was prepared by adding 4.644 g of NAD, and 3.160 g of sodium glucose-6-phosphate (G6P) to the R1 diluent. The powder was completely dissolved by gentle mixing and the pH was adjusted to 7.0 with 10 M NaOH or 3 M HCl. The solution was added to a graduated cylinder and additional R1 diluent was added to give a final volume of 100 mL. This solution was mixed well by gently rotating on a roller and filtered through a 0.2 μm cellulose nitride filter unit.

A 4X antibody working solution (25 ml) was prepared by pre-diluting the antibody stock (6.6 mg/ml) 1:10 fold in R1 diluent to give an antibody solution of 660 μg/ml. 1.59 ml of pre-diluted antibody solution (660 μg/ml) was added to 23.41 ml of R1 diluent to make a 42 μg/ml concentration antibody working solution (4X).

The R1 reagent was prepared by mixing 40 ml of substrate working solution (2X), 20 ml of antibody working solution (4X), and 20 ml of R1 diluent. The solution was mixed gently, covered with aluminum foil, and stored at 4° C. before use.

The R1 blank was prepared by mixing 40 ml of substrate working solution (2X) and 40 ml of R1 diluent. The solution was mixed gently, covered with aluminum foil, and stored at 4° C. before use.

Preparation of R2 reagent and R2 blank

R2 reagent contained SDMA-G6PDH conjugate (0.42 μg/ml) in R2 diluent. R2 blank contained SDMA-G6PDH conjugate (0.014 μg/mL) in R2 diluent.

0.3 ml of conjugate stock (Lot #5616-80-2, 770 μg/ml) was added to 29.7 ml of R2 Diluent to pre-dilute the conjugate 1:100 fold to give a final concentration of 7.7 μg/ml. .

R2 reagent was prepared by adding 21.8 ml of the pre-diluted conjugate solution to 378.2 ml of R2 diluent.

An R2 blank was prepared by adding 0.73 ml of the pre-diluted conjugate solution to 399.27 ml of R2 diluent.

The solution was mixed well and stored at 4° C. before use.

Example 5: Preparation of the SDMA analogue N,N'-dimethylarginine thiol (SDMA-SH)

matter:

- Cysteamine 4-methoxytrityl resin: 0.7 mmol/g resin loading and 200-400 mesh copolymer matrix (Novabiochem).

- Fmoc-SDMA(Boc)2ONa: MW 624.7, purity≥95%, (Novabiochem)

- HATU: MW 380.3 (Novabiochem).

- N,N-diisopropyl ethylamine: 99.5% from Sigma, purified by double distillation.

- DMF (anhydrous): purity≥99.8% from Sigma.

- Piperidine: 20% in anhydrous DMF.

step:

In a 20 mL vial, cysteamine 4-methoxytrityl resin (1.2 g, 0.46 mmol), Fmoc-SDMA(Boc)2ONa (0.9 g, 1.4 mmol, 3.0 equiv), HATU (0.54 g, 1.4 mmol, 3.0 equiv) ), diisopropyl ethylamine (0.4 mL, 2.3 mmol, 5.0 equiv) and anhydrous DMF (18 mL) were added. The mixture was capped and inverted at room temperature for 16 hours. The liquid was removed from the vial using a glass pipette. The resin was then washed with DMF (18 mL, 4 times) followed by methanol (18 mL, 4 times). The resin was inverted in 20% piperidine in DMF at room temperature for 15 minutes (18 mL, 3 times). The resin was then washed with DMF (18 mL, 4 times) followed by methanol (18 mL, 4 times) and dried in a lyophilizer for 1 hour. The yellow/light pink resin can then be stored at 4°C or cleaved to provide SDMA-SH.

SDMA-SH was cleaved from the resin (50 mg) by inversion in trifluoroacetic acid (3 mL) at room temperature for 1 hour. The dark red slurry was then filtered and washed with acetonitrile (1 mL) to give a clear yellow solution. The solution was then lyophilized to give SDMA-SH (7 mg) as a dark yellow oil. Due to oxidation of the thiol, the compound must be used immediately or stored at -80°C (storage at -80°C results in <10% oxidation after 2 months). SDMA-SH (Formula 1) was confirmed by LC/MS and NMR.

Formula 1

Example 6: Conjugation of SDMA and G6PDH

Conjugates of SDMA and G6PDH were prepared by conjugating the SDMA analogue SDMA-SH with G6PDH activated with SIA, SBPA or SMCC in the presence of NAD and G6P. The linker length between SDMA and G6PDH could be varied by selecting the reagents used for activation as shown in Figures 2, 3 and 4.

Enzyme pre-activation by SIA: One vial of glucose-6-phosphate dehydrogenase (G6PDH) (12 mg) was dissolved in 3 ml MES buffer (50 mM, pH 8.0), to ensure complete dissolution of the enzyme rotated for 1 hour. The enzyme solution was kept on ice until needed. An additional 4.5 ml MES buffer (50 mM, pH 8.0) was added to the enzyme solution, mixed well via vortexing (5 sec) and kept on ice for 10 min. 100 mg G6P was dissolved in 1 ml deionized water and kept on ice for 10 minutes. 200 mg NADH was dissolved in 1 ml deionized water and kept on ice for 10 minutes. 0.68 ml G6P solution and 0.34 ml NADH solution were added to the enzyme solution, mixed well via vortexing (5 seconds) and kept on ice for 10 minutes. One vial of SIA (50 mg) was dissolved in 0.5 ml DMSO (100 mg/ml). 0.14 ml of SIA solution was added to the enzyme solution, mixed well via vortexing (5 seconds), covered with aluminum foil, and rotated at room temperature for 2 hours. The solution was transferred to a G2 Slide-A-Lyzer dialysis cassette and dialyzed against PBS buffer (4 L) at 4° C. in the dark for 5 hours. The buffer was replaced with fresh PBS (4 L) and the solution was dialyzed at 4° C. overnight in the dark. The dialysis buffer was replaced with MES (4 L, 25 mM, pH 8.0) and the solution was dialyzed for 3 hours at 4°C. 12.5 ml of enzyme solution was removed from the dialysis cassette and 0.32 ml MES buffer (1 M, pH 8.0) and 0.32 ml EDTA (0.2 M, pH 8.0) were added to obtain a final solution concentration of 50 mM MES and 5 mM EDTA. made it If necessary, if the enzyme solution is less than 12.5 ml, the volume of MES and EDTA can be adjusted accordingly. The solution was degassed with argon for 5 minutes.

Enzyme pre-activation by SBPA: To 2 mg of G6PDH in 1 ml MES buffer (50 mM, pH 8.0), 11.3 mg G6P and 16.8 mg NADH were added, mixed well and kept on ice for 10 minutes. SBPA (50 mg) was dissolved in 0.5 ml DMSO (100 mg/ml). 0.025 ml SBAP was added to the enzyme solution, mixed well via vortexing (5 sec), covered with aluminum foil, and spun at room temperature for 2 h. The solution was transferred to a G2 Slide-A-Lyzer dialysis cassette and dialyzed against PBS buffer (2 L) at 4° C. in the dark for 5 hours. The buffer was replaced with fresh PBS (2 L) and the solution was dialyzed at 4° C. overnight in the dark. The dialysis buffer was replaced with MES (2 L, 25 mM, pH 8.0) and the solution was dialyzed for 3 hours at 4°C. 2.5 ml of the enzyme solution was removed from the dialysis cassette and 0.060 ml MES buffer (1 M, pH 8.0) and 0.025 ml EDTA (0.5 M, pH 8.0) were added to obtain a final concentration of 50 mM MES and 5 mM EDTA. made it become

Enzyme preactivation by SMCC: To 2 mg of G6PDH in 1 ml MES buffer (50 mM, pH 8.0), 11.3 mg G6P and 16.8 mg NADH were added, mixed well and kept on ice for 10 minutes. 50 mg sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was dissolved in 0.5 ml DMSO (100 mg/ml). 0.035 ml SMCC solution was added to the enzyme solution, mixed well via vortexing (5 seconds), covered with aluminum foil, and spun at room temperature for 2 hours. The solution was transferred to a G2 Slide-A-Lyzer dialysis cassette and dialyzed against PBS buffer (2 L) at 4° C. in the dark for 5 hours. The buffer was replaced with fresh PBS (2 L) and the solution was dialyzed at 4° C. overnight in the dark. The dialysis buffer was replaced with MES (2 L, 25 mM, pH 8.0) for 3 hours at 4°C. 2.5 ml of the enzyme solution was removed from the dialysis cassette and 0.060 ml MES buffer (1 M, pH 8.0) and 0.025 ml EDTA (0.5 M, pH 8.0) were added to obtain a final concentration of 50 mM MES and 5 mM EDTA. made it become

Conjugation of SDMA and preactivated G6PDH: Freshly prepared SDMA-SH (Formula 1) was added to the preactivated enzyme solution in the following amounts: 0.35 ml (100 mg/ml) for SIA-activated G6PDH and SBAP or 0.065 ml (100 mg/ml) for SMCC-activated G6PDH. The solution was mixed well and the mixture was rotated at 4° C. for 36 hours. The reaction mixture was dialyzed (3 or 4 cycles) at 4° C. against PBS (2 or 4 L) using a G2 Slide-A-Lyzer dialysis cassette (Thermo Scientific). The SDMA-enzyme conjugate solution was equilibrated by dialysis against Tris HCl buffer (25 mM, pH 8.0) for 4 hours at 4°C. The solution was filtered using a 0.45 μm centrifugal filter (1500*g for 10 minutes).

Table 5 shows the linker length and activity of each conjugate and the ability of the antibody to inhibit G6PDH of the conjugate. The linker length does not include non-natural nitrogen in the SDMA derivative of Formula 1. Preactivation of G6PDH in the presence of SIA resulted in better enzymatic activity than conjugates prepared with SBPA or SMCC.

table 5

Example 7: Preparation of anti-SDMA monoclonal antibodies

Methods of producing monoclonal antibodies are within the skill of the art. In one embodiment, the antibody is a monoclonal antibody raised against the SDMA-KLH conjugate having the structure:

Purification of anti-SDMA antibodies was performed as follows:

matter:

2 x 2L of anti-SDMA

Reverse-phase (rPA) column (5ml) dedicated to anti-SDMA IgG purification (GE Healthcare)

· Pierce IgG Purification Buffer

· IgG binding buffer

IgG low pH elution buffer

· Dialysis PBS, pH 7.4

2 x 4L @ 4C

protocol:

The rPA column (5 ml) was equilibrated in 3 ml/min binding buffer

1. Flow monitored by OD 280 nm

1000 ml of anti-SDMA was diluted in an equal volume of Pierce IgG Binding Buffer

1. The process is repeated for an additional 1000 ml

Diluted anti-SDMA loaded at 6 ml/min onto rPA column

1. OD monitored by 280 nM

The rPA column was washed with PBS/IgG Binding Buffer 1:1 @ 3ml/min until...

1. OD 280 nM reached baseline

Anti-SDMA IgG was eluted using IgG low pH elution buffer @ 3 min/ml

1. Peaks were collected manually

Anti-SDMA IgG was immediately dialyzed against 2 changes of 4 L PBS

1. 30 ml 10k MWCO Cassette(s)

2. Pool the volumes and determine the IgG concentration using OD 280 nm

Antibodies were analyzed by SDS Page, SEC and plate-based immunoassays as described in US Pat. No. 8,481,690.

Example 8: Assay procedure

A modified assay of the present disclosure was performed for SDMA in canine, feline and human samples.

Reagent components are presented in Table 5.

table 5

Pipetting volumes for reagents prepared as described above were the same for color assay and blank assay:

Volume (μl)

Sample or Calibrator 10

R1 40

R2 125

Color and blank assays and related calculations for samples and calibrators were performed on a Beckman AU680® automated analyzer. Calibration standards containing 56.0, 28.0, 14.0, 7.0, and 3.75 and 0 μg/dL of SDMA in the calibrator matrix (removed dog serum) were used for both cats and dogs. The analyzer was programmed to perform color and blank assays as follows: Samples and calibrators were added to reagent R1 and incubated for 3-4 minutes prior to addition of R2. OD was measured at 340 nm using a start at about 36 seconds after addition of R2. Absorbance was measured every 18 sec for 4 additional 18 sec cycles. The absorbance values of the calibrator matrix (0 μg/dL SDMA) were subtracted from each measurement used to calculate the response rate (change in absorbance/minute) of the color and blank assays to give normalized color and blank response rates.

To determine the rate for the standard curve, the normalized rate for each calibrator in the blank assay was subtracted from the normalized rate for each calibrator in the color assay. The sample SDMA concentration was determined by subtracting the normalized rate for the sample in the blank assay from the normalized rate for the sample in the color assay to give the final sample reaction rate, which was compared to the final standard curve.

Table 6 presents the results of the SDMA assay for cat and dog sera when using the color assay alone ("no subtraction") and when the blank assay rate was subtracted from the color assay rate ("with subtraction"). Sample bias reflects the difference between LC/MS results and results using the above procedure.

table 6

Table 7 presents calibration curves prepared with and without subtraction using known amounts of SDMA in stripped dog serum. Similar curves were made for other species.

table 7

Table 8 presents the values for the calibration curve shown in Figure 6 using uncleared human serum spiked with SDMA using the rate assay procedure described above (ie, subtracted color and blank assays).

Table 8

The curves were used to determine concentrations in normal and chronic kidney disease human serum samples as shown in FIG. 1 .

Example 9: Use of Enzyme Inhibitors

The lactate dehydrogenase inhibitor sodium oxamate was added to the blank reagent diluent and reagents as described above in Example 3. The use of inhibitors can improve the accuracy of the assay as shown in Table 9.

table 9

In this example, standard curves for dog serum were made with and without sodium oxamate as shown in Table 10. Similar curves can be prepared for other species.

Table 10

Example 10: Testing human serum samples with stripped and non-cleared human serum calibrators

Endogenous and charcoal-depleted human serum was used as a calibrator matrix to determine the recovery of SDMA in human serum samples. Using the data collected in this experiment, a calibration curve can be created using the speed and fixed calibration methods.

Assay reagents were prepared as shown in Table 11.

Table 11

Anti-SDMA mAb was prepared as in Example 7 and used at an assay concentration of 1.5 μg/mL.

SDMA-G6PDH was prepared as in Example 6 and used at an assay concentration of 0.3 μg/mL.

Calibrators were prepared with decharred human serum with the following SDMA concentrations (μg/dL): 0.0, 4.7, 15.0, 29.0, 59.0 and 111.0 as determined by LC/MS.

The rate method was performed according to Example 8.

In the stationary method, the Beckman instrument was set to measure the change in absorbance at 340 nm between 1 and 3 minutes after initiation of the reaction.

The results of the fixed and rate calculation method for determination of SDMA concentration are shown in FIG. 7 .

Example 11: Human Calibrator Capacity Not Cleared by Buffer Calibrator

The SDMA assay was calibrated using a buffer-based calibrator (0, 6, 11, 24, 46, and 95 μg/dL SDMA in PBS buffer with 1% BSA) and unremoved human serum standards to determine recovery. was used for testing. This experiment used a fixed method to calculate the change in absorbance at 340 nm in machine cycles 12 and 16, each cycle being 18 seconds (T1 = 216 seconds, T2 = 288 seconds). The absorbance from the reagent blank (diH2O) was not subtracted from the net absorbance.

The assay was run as set forth in Example 9. The results are shown in FIG. 8 .

Example 12: Comparison of manual and automated background subtraction

Automated background subtraction method An on-board Beckman AU680® analyzer was tested to determine the effectiveness of the on-board solution. The SDMA assay was run according to the onboard instructions and the assay was run separately and results were calculated using the offline method as a control.

A decharred dog serum calibrator was prepared as in Example 1. Reagent components are presented in Table 12.

Table 12

The reaction volume was:

Sample volume: 17 μL

Reagent 1 volume: 25 μL

Reagent 2 volume: 125 μL

Color and blank assays are not coupled to the on-board analyzer. Data are generated separately for each assay and processed on the machine. The blank assay reaction OD is subtracted from the color assay reaction OD for the calibrator and sample. A curve is fitted to the calibration data and the sample concentration is determined from the OD of the reaction subtracted using the equation of the best fit curve.

Table 13 presents the net change in absorbance for the calibrators in the fixed reaction method.

Table 13

Table 14 shows the measurement of SDMA in the samples using a standard curve generated from the calibrator presented in Table 13.

Table 14

Example 12: Analysis of calibrator matrices from different species

A set of calibrators prepared from pooled sera of various animal species was analyzed to determine the robustness of the SDMA assay when using calibrators with sera from different species.

Assays for human SDMA were performed on calibrators prepared with depleted or endogenous (non-depleted) serum from dogs, cats, and horses using the fixation method as in Example 10. The assay results are shown in Figures 8, 9 and 10.

Example 13: Buffer-Based Calibration

Calibration curves were generated for the calibration concentrations in Table 13 using 1% BSA in PBS as a calibration matrix. Results are shown in FIG. 12 .

Example 14: SDMA assay with no conjugate added to blank reagent

The SDMA assay was run with the reagent concentrations in Table 5 using the rate procedure of Example 8, except that no conjugate was added to R2. The assay was run on a Beckman analyzer. Results are presented in Table 15.

Table 15

Although various specific embodiments of this disclosure have been described herein, it is understood that this disclosure is not limited to such precise embodiments and that various changes or modifications may be made therein by those skilled in the art without departing from the scope and spirit of this disclosure. It should be.

The examples given above are illustrative only and are not meant to be an exhaustive list of all possible embodiments, applications or variations of the present disclosure. Accordingly, various modifications and variations of the described methods and systems of this disclosure will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Although the present disclosure has been described with respect to specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to those specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure obvious to those skilled in molecular biology, immunology, chemistry, biochemistry or related fields are intended to be within the scope of the appended claims.

Any numerical value recited herein includes all values from the lower value to the higher value in increments of one unit, provided there is a separation of at least two units between any lower value and any higher value. . As an example, it is stated that the concentration of a component or the value of a process variable such as, for example, size, angular size, pressure, time, etc. is, for example, 1 to 90, particularly 20 to 80, more particularly 30 to 70 In this case, values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc. are intended to be expressly recited herein. For values less than 1, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest and highest values enumerated are to be regarded as expressly recited herein in a similar manner.

Claims (80)

A conjugate comprising symmetric dimethyl arginine (SDMA) conjugated to glucose-6-phosphate dehydrogenase (G6PDH), wherein SDMA is conjugated to G6PDH via a 5-9 member linker. delete 2. The conjugate of claim 1, wherein SDMA is conjugated to G6PDH via a linker having a length of 5-15 Angstroms. The conjugate of claim 1 selected from the group consisting of:

, and

. A composition comprising the conjugate of claim 1 and an antibody specific for free SDMA. 6. The composition of claim 5, wherein the antibody specific for free SDMA has a reactivity to asymmetric dimethylarginine (ADMA) that is less than 25% of its reactivity to free SDMA. 7. The composition of claim 6, wherein the antibody has no or substantially no cross-reactivity with one or more compounds selected from the group consisting of asymmetric dimethylarginine (ADMA), L-arginine, and N-methylarginine. A kit comprising components of the conjugate of claim 1 and an antibody specific for SDMA. 9. The kit of claim 8, further comprising a component of nicotinamide adenine dinucleotide (NAD). A reaction mixture comprising the components of the kit of claim 8 and a sample suspected of containing SDMA. (a)i. A first reagent comprising an anti-analyte antibody and a signal generating substrate for G6PDH, and
ii. A first for performing a first assay comprising an analyte and a second reagent comprising a conjugate of G6PDH, wherein the conjugate comprises SDMA conjugated to G6PDH via a 5-9 member linker. a set of reagents, and
(b) i. a second set of reagents for performing a second assay comprising a third reagent comprising a substrate;
A kit containing reagents for performing an assay on a sample containing an analyte. 12. The kit of claim 11, wherein the second set of reagents further comprises a fourth reagent comprising at least one of a diluent and a buffer. 13. The kit of claim 12, wherein the fourth reagent further comprises the conjugate or G6PDH. 13. The kit of claim 12, wherein at least one of the reagents comprises an inhibitor to an enzyme other than G6PDH. 13. The kit of claim 12, wherein the concentration of the conjugate of the first reagent is 5 to 150 times higher than the concentration of the conjugate of the fourth reagent. 12. The kit of claim 11, further comprising a standard comprising a known amount of the analyte diluted in the analyte-free sample solution. 17. The kit according to claim 16, wherein the analyte-free sample solution is a stripped-serum, stripped-plasma, or pre-treated sample. 12. The method of claim 11, wherein at least one of the first reagent and the second reagent further comprises an electron mediator and a dye, and the third reagent further comprises a mediator and a dye, wherein the mediator accepts electrons from the substrate and converts it to a dye. Kit to move. 19. The method of claim 18, wherein at least one of the first reagent and the second reagent comprises an electron mediator and a dye, and at least one of the third reagent and one of the fourth reagent comprises a mediator and a dye, wherein the mediator comprises an electron mediator and a dye. A kit that receives and transfers it to a dye. 12. The kit of claim 11, wherein the analyte is free symmetric dimethyl arginine (SDMA). 21. The kit of claim 20, wherein the anti-analyte antibody is an antibody specific for free SDMA. 22. The kit of claim 21, wherein the antibody has a reactivity to asymmetric dimethylarginine (ADMA) that is less than 25% of its reactivity to free SDMA. 23. The kit of claim 22, wherein the anti-analyte antibody has no or substantially no reactivity to ADMA, L-arginine, and N-methylarginine. delete 12. The kit of claim 11, wherein the conjugate comprises SDMA conjugated to G6PDH by a linker having a length of 5 to 15 Angstroms. 12. The kit of claim 11, wherein the substrate comprises NAD. 12. The kit of claim 11, wherein the second reagent further comprises glucose-6 phosphate (G6P). (a) contacting the sample with a substrate comprising an anti-SDMA antibody specific for free SDMA, the conjugate of claim 1, and NAD;
(b) measuring the conversion of NAD to NADH, and
(c) determining the presence or amount of SDMA in the sample based on the conversion of NAD to NADH.
A method for determining the presence or amount of free SDMA in a sample. 29. The method of claim 28, wherein measuring the conversion of NAD to NADH comprises measuring the conversion of NAD to NADH. 30. The method of claim 29, wherein determining the presence or amount of SDMA in the sample based on NAD to NADH conversion comprises comparing the NAD to NADH conversion to a standard curve. 29. The method of claim 28, wherein measuring the conversion of NAD to NADH comprises measuring the amount of conversion of NAD to NADH. 32. The method of claim 31, wherein determining the presence or amount of SDMA in the sample based on NAD to NADH conversion comprises comparing the amount of NAD to NADH conversion to a standard curve. 29. The method of claim 28, wherein the sample is serum, plasma, urine or cerebrospinal fluid. 29. The method of claim 28, wherein the conjugate is selected from the group consisting of:

, and

. 29. The method of claim 28, wherein the anti-SDMA antibody specific for free SDMA has a reactivity to asymmetric dimethylarginine (ADMA) that is less than 25% of its reactivity to free SDMA. 36. The method of claim 35, wherein the anti-SDMA antibody has no or substantially no reactivity to ADMA, L-arginine, and N-methylarginine. delete 29. The method of claim 28, wherein SDMA is conjugated to G6PDH via a linker having a length of 5 to 15 Angstroms. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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